1. Field of the Invention
The invention is in the field of antibody-based assays for toxins having peptidase activity. In particular, this invention relates to assays for toxins, in particular botulinum neurotoxins and tetanus toxins. The invention also relates to antibodies useful in the assays and to peptides immobilized on solid phase supports that are useful in the assays.
2. Related Art
The botulinum neurotoxins are a family of structurally similar, but antigenically different protein neurotoxins which act on the peripheral nervous system to block neuromuscular transmission. These neurotoxins are extremely potent, with a human lethal dose in the order of micrograms, and give rise to the rare but frequently fatal disease, botulism. Assays for the botulinum neurotoxins are currently used in both the food and pharmaceutical industry. The food industry employs assays for the botulinum neurotoxins to validate new food packaging methods and to ensure food safety. With the growing clinical use of the botulinum toxins, the pharmaceutical industry requires accurate assays for these toxins for both product formulation and quality control.
It is known to assay for botulinum toxin in foodstuffs using the mouse lethality test. This test has been the industry standard for many years, though over the past 10 years a number of immunoassay methods have been developed in an attempt to replace the mouse test in the majority of applications.
One such assay operates by addition of a test sample to a plate or column to which is attached an antibody that binds to toxin present in the sample. A further antibody is typically used to detect bound toxin. These enzyme-linked immunoassays (ELISA) have the advantages that they are specific to one botulinum toxin type and can be performed rapidly, in less than 2 hours. The ELISAs, however, suffer from several drawbacks:
(a) They do not measure the biological activity of the toxins,
(b) They cannot distinguish between active and inactive toxin, and
(c) Due to antigenic variations some toxins are not detected by these assays which therefore give rise to false negatives.
The botulinum neurotoxins have recently been shown to possess highly specific zinc-endopeptidase activities within their light sub-units. Depending on the neurotoxin type these act to cleave small proteins within the nerve cell which are involved in neurotransmitter release. Botulinum types A and E toxins cleave protein SNAP-25. Botulinum types B, D, F and G and tetanus toxins cleave vesicle-associated membrane protein (VAMP-also called synaptobrevin). Botulinum type C toxin cleaves the protein syntaxin.
In the development of further toxin assays, various procedures have been devised for the evaluation of endopeptidase activities. Liquid chromatography procedures are known and are based on resolution of the peptide product and subsequent evaluation. These procedures are time-consuming, expensive and do not lend themselves readily to automation. It is also known to use spectrophotometric methods, requiring the development of suitable chromogenic peptide reagents. Such methods provide a continuous precise assay for endopeptidases. Spectrophotometric methods, however, require relatively pure preparations of enzyme and are not normally suitable for evaluation of endopeptidase activities in crude or particulate samples.
Despite these efforts, at present, the only convenient assay for the biological activity of the botulinum neurotoxins, and the only assay that is FDA approved, remains the mouse lethality test. This test suffers from a number of drawbacks:
(a) It is expensive and uses large numbers of laboratory animals,
(b) It is non-specific unless performed in parallel with toxin neutralization tests using specific anti-sera, and
(c) It is not very accurate unless large animal groups are used.
The present invention describes a novel assay system for toxins, using novel reagents. The assay aims to overcome or at least mitigate many of the drawbacks of present in vitro assays for these toxins.
The invention relates to an assay for botulinum toxin or tetanus toxin comprising the steps of:
(a) combining a test compound with a substrate and with antibody, wherein the substrate has a cleavage site for the toxin and when cleaved by toxin forms a product, and wherein the antibody binds to the product but not to the substrate; and
(b) testing for the presence of antibody bound to the product, which product is attached to a solid phase assay component.
Preferably, in the practice of this invention, the substrate is a peptide or a protein which is cleaved by the toxin to generate new peptides having N- and C-terminal ends. In addition, the peptide substrate is attached to a solid phase component of the assay.
The assay according to the invention may utilize assay components (a) and (b):
(a) a peptide linked to a solid-phase, the peptide being cleavable by the toxin to generate a cleavage product,
(b) an antibody that binds to the cleavage product but not to the peptide, and the assay may comprise the steps of:
(i) combining a test compound that may contain or consist of the toxin with the solid-phase peptide to form an assay mixture,
(ii) subsequently or simultaneously combining the assay mixture with the antibody, and
(iii) subsequently or simultaneously determining whether there has been formed any conjugate between the antibody and the cleavage product.
Preferably, the step (i) of the assay is carried out in the presence of a zinc compound. In addition, the peptide is selected from intact peptides or fragments thereof selected from the group consisting of VAMP; a VAMP analog; a VAMP isoform; SNAP-25; a SNAP-25 analog; a SNAP-25 isoform; syntaxin; a syntaxin analog; and a syntaxin isoform; or a fragment thereof.
In this embodiment, the assay comprises:
(i) combining the test compound with a solid phase comprising a peptide selected from the group consisting of VAMP; a VAMP analog; a VAMP isoform; SNAP-25; a SNAP-25 analog; a SNAP-25 isoform; syntaxin; a syntaxin analog; and a syntaxin isoform; or a fragment thereof,
(ii) washing the test compound from the solid phase,
(iii) combining the solid phase with an antibody adapted for binding selectively with peptide cleaved by toxin, and
(iv) detecting a conjugate of the antibody with cleaved peptide.
In another embodiment, the assay comprises:
(i) adding a test solution to an assay plate comprising immobilized peptide, the peptide being selected from the group consisting of VAMP; a VAMP analog; a VAMP isoform; SNAP-25; a SNAP-25 analog; a SNAP-25 isoform; syntaxin; a syntaxin analog; and a syntaxin isoform; or a fragment thereof,
(ii) incubating the assay plate,
(iii) washing the plate with a buffer,
(iv) adding to the plate an antibody solution, said solution comprising an antibody adapted selectively to bind to a peptide selected from the group consisting of (1) a peptide the C-terminal end of which is selected from the group consisting of SEQ ID NOS: 1, 3 and 5, and (2) a peptide the N-terminal end of which is selected from the group consisting of SEQ ID NOS: 2, 4 and 6,
(v) incubating the assay plate,
(vi) washing the plate with a buffer, and
(vii) measuring the presence of antibody on the assay plate.
In this embodiment, the antibody may be linked to an enzyme and the presence of antibody on the plate is measured by adding an enzyme substrate and measuring the conversion of the substrate into detectable product. The detectable product may be colored and measured by absorbance at a selected wavelength.
In the practice of the invention, the inactive toxin present in the test compound may be converted to active toxin. This may be accomplished by adding a protease to the test compound.
The antibody-peptide conjugate may be detected using a further antibody specific to the first antibody and linked to an enzyme.
The present invention also relates to a method of obtaining an antibody, the antibody being for use in an assay for botulinum toxin or tetanus toxin, the method comprising identifying a macromolecule that is cleaved by the toxin into cleavage products, immunizing an animal against one of the cleavage products, isolating antibodies that bind to said one of the cleavage products and recovering antibody that does not cross-react with the macromolecule. In this embodiment, the method comprises:
(i) identifying a macromolecule that is cleaved by the toxin, thereby forming at least first and second cleavage products,
(ii) immunizing an animal with a selected one of the cleavage products,
(iii) isolating from the animal an antibody that binds to the selected cleavage product, and
(iv) checking the antibody does not bind to the macromolecule.
Preferably, the method comprises:
(i) immunizing an animal with an antigen selected from the group consisting of (1) SEQ ID NO.s 1-6; and (2) a carrier molecule linked to (1), and
(ii) isolating from the animal an antibody that binds to the antigen.
In the practice of this aspect of the invention, the antigen may be linked to a carrier protein, e.g. selected from Keyhole Limpet Hemocyanin, Bovine Serum Albumin and Ovalbumin.
The invention also relates to an antibody for use in the assay according to the invention, characterized in that it does not bind to a peptide selected from the group consisting of VAMP; a VAMP analog; a VAMP isoform; SNAP-25; a SNAP-25 analog; a SNAP-25 isoform; syntaxin; a syntaxin analog; and a syntaxin isoform; or a fragment thereof; but does bind to a product of cleavage by the toxin of one of these peptides. Preferably, the antibody is adapted to bind to an antigen of formula:
Pxe2x80x94Qxe2x80x94X
wherein X is a peptide selected from the group consisting of SEQ ID NOS: 1-7 and is covalently linked at one end to P-Q, and wherein P-Q is a carrier in which P is a carrier protein and Q is an amino acid or an amino acid sequence adapted to attach peptide X to carrier P.
The invention also relates to a component for a toxin assay comprising a peptide selected from the group consisting of VAMP; a VAMP analog; a VAMP isoform; SNAP-25; a SNAP-25 analog; a SNAP-25 isoform; syntaxin; a syntaxin analog; and a syntaxin isoform; or a fragment thereof; and wherein the peptide is immobilized on a solid phase.
The invention also relates to a toxin assay kit comprising
(1) an assay component according to the invention;
(2)
(a) an antibody according to the invention which is linked to or including an enzyme, or
(b) an antibody according to the invention which is unlabeled with an enzyme and a second antibody adapted to bind to the first antibody and linked to or including an enzyme, and
(3) a substrate for the enzyme.